Apparatus for embedding electron microscopy tissue

ABSTRACT

Tissue particles intended to be thinly sliced for electron microscopy examination are placed in capsules filled with an epoxy resin embedding mixture and the capsules are received by a holder which is both vibrated and sequentially heated by an apparatus programmed to a time-temperature sequence which apparatus and method drastically reduces embedding time.

United States Patent [1 1 Kinney et al. I

[ Dec. 18, 1973 APPARATUS FOR EMBEDDING ELECTRON MICROSCOPY TISSUE [76] Inventors: Thomas D. Kinney, 3120 Devon Rd.; John E..P. Pickett, 3323 Pinafore Dr., both of Durham, NC.

[22] Filed: July 6, 1971 [21] Appl. No.: 159,998

[52] US. Cl.....-. 425/160, 259/54, 425/432, 425/456 [51] Int. Cl B0lf 11/00 [58] Field of Search 425/160, 432, 456; 259/54 [56] References Cited UNITED STATES PATENTS 2/1970 Rudge 425/[60 X 3,635,446 l/l972 Kurosawa et al. 259/54 FOREIGN PATENTS OR APPLICATIONS l,437,055 3/1966 France 259/54 Primary Examiner-Robert D. Baldwin Attorney-B. B. Olive [57] ABSTRACT Tissue particles intended to be thinly sliced for electron microscopy examination are placed in capsules filled with an epoxy resin embedding mixture and the capsules are received by a holder which is both vibrated and sequentially heated by an apparatus programmed to a time-temperature sequence which apparatus and method drastically reduces embedding time.

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' FIG. 6 20 27 ELow Temperature 13) 1 Vibrator Motor To Power I High Temperature INVENTORS Thomas D. Kinney John E. P. Pickett ATTORNEY APPARATUS FOR EMBEDDING ELECTRON MICROSCOPY TISSUE BACKGROUND OF THE INVENTION 1. Field of the Invention The invention relates broadly to molding but more specifically to apparatus for embedding tissue particles preparatory to thin slicing for electron microscopy examination.

2. Description of the Prior Art Various apparatus and methods have been employed for processing and embedding the minute size tissue which after thin slicing is further processed and used for electron microscope examination. A common practice is to fill a plurality of so-called Beem plastic capsules with a premixed epoxy resin embedding plastic such as the Epon plastic sold by Shell Oil Company. A single piece of tissue in the order of 1 cm is placed in each capsule. The capsules sit in racks at room temperature until each piece of tissue has settled through the epoxy resin plastic to the bottom of its respective capsule. Subsequent cutting procedures require the particles to be at the bottom when the embedding mixture is cured. This submersion and settling operation may require one to two hours and frequently the capsules are left longer, often overnight. Various prodding devices such as needles and forceps are used to force the tissue particles to submerge and settle and it is found that some particles after settling will sometimes tend to rise. Different kinds of particles react differently to settling. Once the particles are seen to be at the bottoms of the respective capsules, the rack of capsules is placed in an oven maintained at 60C for curing the embedding plastic. The capsules are left in the oven until hard and this normally requires from 36 to 48 hours. The capsules are then removed from the oven for cooling and after being cooled the epoxy resin is relatively hard and after having the capsule removed. is ready for electron microscopy microtome cutting. Some researchers use gelatin capsules rather than the Beem type plastic capsules but in either case the process of getting the tissue particle to settle and the epoxy resin to harden is slow and tedious. The increase in both research and patient loads found in most medical centers has magnified the need for reducing the time required to embed electron microscopy tissue. A summary of the technology is found in the booklet Thin Sectioning and Associated Technics for Electron Microscopy, Second Edition with Supplement, published in 1967 by Ivan Sorvall, Inc. of Norwalk, Connecticut, 06852 (see page 22 for a typical Epon mixture which gives the problem). Another reference is the article A Fast Method for Processing Biologic Material for Electron Microscopy" by Sergio A. Bencosme and Victor Tsutsumi in the publication Laboratory Investigation Vol. 23, No. 4, p. 447, 1970 by the International Academy of Pathology. In this last article mention is made of reducing the time for embedding electron microscopy tissue particles in epoxy resin by placing the capsules in two successive ovens at two successive temperatures. This article does not mention the time shown by experience to be required for assisting the particles to submerge. Taking all of the known prior art into account it can be said that the art has not provided an apparatus and method which will rapidly cure epoxy resin type plastic in embedding capsules without requiring assistance in submerging the particles and without requiring handling of the capsules during curing and whose total time for embedding after the capsules are filled and the particles placed approximates four hours compared to 36 to 60 hours with the method and apparatus most commonly used for epoxy resin embedding. In particular, the art has not provided an apparatus and method based on vibrating the electron microscopy plastic filled capsules to assist in submerging the particles and in a program of heating at different temperatures and which does not require that the capsules be moved from one apparatus to another apparatus.

SUMMARY OF THE INVENTION According to the apparatus of the invention a relatively large number of capsules, e.g. the well-known Beem type, are placed in an aluminum block of considerable heat mass. The block is fitted with small wells which fit the capsule shape and one capsule is dropped into each well. The capsules are filled with an epoxy resin plastic, e.g. the Epon type plastic, and a single tissue particle is dropped in each capsule. The block is then placed in an apparatus having a housing which includes a large well adapted to receive the block on a base plate. Surrounding the block at appropriate locations are separate sets of adjustable electrical heaters one set of which is set to operate at a relatively low temperature of 60C and the other of which is set to operate at a relatively high temperature of C. Below the base plate is a vibratory motor adapted to induce vibrations in the base plate and into the block when set on the base plate so as to vibrate the capsules and their contents. The housing also contains a separately powered electrical sequence program switch with appropriate electrical switching and timing means so that the heaters and vibratory motor can be operated in a particular sequence. A single two-step heater may be used.

Once the block is in position in its well in the apparatus and the various capsules are filled with the epoxy resin and the respective tissue particles, the vibratory motor is started for a period of time during which the heaters are off. This vibrates the block and effectively agitates the embedding plastic so that the particles tend to submerge and sink to the bottom of their respective capsules without requiring the effort or time involved with the usual needles, forceps or other prodding devices to assist the particles to submerge. The agitation or vibration stops after a programmed time period and the block is then heated on low temperature for a predetermined length of time. Next, the block is heated at high temperature for a predetermined time period and then the heat cuts completely off. Following a time period allowed for the block and capsules to cool, the capsules are ready to be removed from the block and stripped from the respective now hardened epoxy resin blocks containing the respective particles. The process is continuous and no handling of particles or capsules is required after the vibration is started and until the capsules are cool and ready to remove.

The programmer is of a type which allows variable time settings for the agitation of the capsules, for the low heat and for the high heat. The programmer cuts off automatically and the operator can control the starting position to control the agitation or vibration time. Ready-to-cut plastic embedded tissue can be provided in four to four and one-half hours. Handling of surgical tissues for electron microscopy examination on a large scale basis for diagnosis is now possible. While found specifically useful with epoxy resin embedding mixtures the invention is deemed applicable to any embedding mixture of similar character in viscosity, hardening character and the like.

DESCRIPTION OF THE DRAWINGS FIG. 1 is a plan view of a programmed heat-vibratory apparatus made according to the invention and showing the capsule holder block installed in its well and some of the block wells filled with capsules.

FIG. 2 is a plan view like FIG. 1 but with the block removed.

FIG. 3 is a front elevation view of the apparatus with the capsule holder block installed.

FIG. 4 is a bottom view showing the programmer switch and vibratory motor.

FIG. 5 is a section view through the capsule holder block.

FIG. 6 is a somewhat schematic circuit diagram of the programmer or timer switch, the heater elements and vibrator motor.

DESCRIPTION OF THE PREFERRED EMBODIMENT As shown in the drawings, a housing 10 is provided with a large well 11 having a metal base plate below which is secured an electric vibrator motor 13. Motor 13 is provided with a small off center weight 14 whose rotation causes a small vibration in base plate 12. Any small fractional horsepower motor such as used for record players, timers and the like appears adequate. A heavy aluminum block 15 is provided with a plurality of smaller wells 16 which are shaped to receive the Beem type capsule 17. Housing 10 also mounts an electrical sequence programmer or timer switch 20 having a power supply cord 21. While a wide variety of program switches suited to the invention are available one model is the Industrial Sequence Programmer" made by the Mallary Timer Company, 3020 East Washington Street of Indianapolis, Indiana, 46206. This switch has its own motor and is available for 125 volt, 60 cycle operation and in use is connected to provide power and timing to operate the vibrator motor 13, an adjustable module heater for low heat operation, an adjustable module heater 26 for high heat operation, a low heat signal light 27 and a high heat signal light 28. It should be understood that the respective heaters 25 and 26 while shown only in FIG. 6 should be understood as being suitably placed adjacent the capsule holder block 15 to provide the necessary heat.

Switch 20 is controlled by a knob 30 and the respective knobs 31 and 32 control the fine adjustments for low heat and high heat respectively. Sequence programmer switches of the type mentioned act both as on-off switches as well as timing switches. In the preferred apparatus of the invention the programmmer is set so that just past an arbitrary off position the timing cycle starts which controls operation of vibrator motor 13. As the timing switch 20 turns in the customary manner and under the power of its own drive motor, the vibratory motor will remain on and will cause base plate 13 to vibrate and accordingly will cause the filled capsules 17 to be agitated so that the particles are assisted in submerging as required. This phase of the timing has been found to require one and one-half hours after which all of the particles in a block having twenty Beem type capsules were found to have become properly positioned. This time can be shortened either by appropriate adjustment of the timing cams of switch 20 or simply by moving control knob to a later starting position which cuts down the time available for vibration and agitation of the capsules 17. Switch 20 then causes vibrator motor 20 to cut off and there is then a time delay of approximately 20 minutes which allows the now somewhat fluid plastic in the capsules to settle. Following this delay switch 20 causes the low temperature heater 25 to be energized as indicated by signal light 27 and the capsule holder block 15 is heated for one and one-half hours at 60C. Switch 20 as it continues to turn then cuts off the low temperature heater 25 and cuts on the high temperature heater 26 which heats the capsule holder block 15 at C for one and onehalf hours. The high temperature heater 26 is then cut off and as the switch 20 rotates back to its zero and stop position over a period of another hour, the capsules are left to cool with no vibration and no heat. Where fine temperature adjustments are desired to vary the respective low and high temperature settings the adjusting knobs 31 and 32 are used for this purpose.

What is particularly significant about the present invention is that with the advent of new developments which substantially reduce the time required to process the electron microscopy tissue particles prior to embedding in the epoxy resin for cutting it now becomes possible to process, embed and cut electron microscopy tissue particles in one day which cuts off three to four days normally required for these steps. Of equal significance, it is beleived, is the fact that the present apparatus completley eliminates the need to prod and poke at the tissue particles during embedding to make them submerge thus the risk of tissue damage from this operation has been eliminated. Very excellent, well cured specimens have been achieved in every instance. While particularly directed to epoxy resin embedding mixtures such as the Epon mixture, the invention readily adapts to any embedding mixture of similar character with regard to the tissue settling and hardening requirements. Also, the heaters (25, 26) may constitute a single adjustable heater.

What is claimed is:

1. An apparatus for embedding electron microscopy tissue particles, comprising in combination:

a. a housing having formed therein an open top recessed well having a bottom base plate and interior side walls surrounding and above said plate and having a housing top wall surrounding said well;

b. electrically operable vibratory means mounted in said housing proximate said plate and adapted when energized to vibrate said plate;

c. first adjustable electrical heater means positioned proximate said well having a rotatable control mounted on said top wall and adapted when energized to heat said well at some predetermined first embedding curing temperature of about 60C;

(I. second adjustable electrical heater means positioned proximate said well having a rotatable control mounted on said top wall and adapted when energized to heat said well at some predetermined second embedding curing temperature of about 85 e. signal light means mounted on said top wall and wired to indicate respective energizations of said first and second heater means;

f. a rotatable electric timer switch adapted when rotated to an on position to thereafter automatically rotate and in turn energize first only said vibrator means for a predetermined first time period of about one and one-half hours, then only said first heater means for a predetermined second time period of about one and one-half hours, then only said second heater means for a predetermined third time period of about one and one-half hours; and whereby each said capsule and its respective mixture then to an off position; and 10 and particle may be successively vibrated for said first g. a metal block of relatively large heat mass adapted time period, then heated at a relatively low temperato fit in said well and rest on said plate and in seture for said second time period, then heated at a relaquence to be vibrated for said first time period, to tively higher temperature for said third time period and next be heated in said well at said first temperature then cooled and hardened while resting on said block for said second time period, then to be heated in thereby allowing the respective particles to be gradusaid well at said second temperature for said third ally submerged to the bottom of the respective capsules time period, and then to cool while said switch reand encased in the hardened mixture therein. mains in said off position, said block having a plu- 

